History of Discovery
Smooth Muscle Phenotypic Modulation—
A Personal Experience
Julie H. Campbell, Gordon R. Campbell
Abstract—The idea that smooth muscle cells can exist in multiple phenotypic states depending on the functional demands
placed upon them has been around for >5 decades. However, much of the literature today refers to only recent articles,
giving the impression that it is a new idea. At the same time, the current trend is to delve deeper and deeper into
transcriptional regulation of smooth muscle genes, and much of the work describing the change in biology of the cells
in the different phenotypic states does not appear to be known. This loss of historical perspective regarding the biology
of smooth muscle phenotypic modulation is what the current article has tried to mitigate. (Arterioscler Thromb Vasc
Biol. 2012;32:1784-1789.)
Key Words: contractile state ◼ synthetic state ◼ smooth muscle differentiation
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I
n 1968, Robert Wissler1 suggested that the cells of the arterial media are of prime importance in the pathogenesis
of atherosclerosis and that they comprise a single cell type.
Before this time, most histology textbooks stated that both
smooth muscle and fibroblasts existed in the media of medium
and large arteries. He further noted that these cells, which
maintained vascular tone and thus were contractile, were also
responsible for the production of collagen and elastin. Wissler
thus proposed that the arterial medial cell could be viewed as a
multifunctional mesenchyme, that the migration and proliferation of medial smooth muscle cells are major cellular sources
of thickened neointimas, and that many of the so-called lipidladen foam cells in atherosclerotic lesions may be modified
smooth muscle cells.
whole blood serum. Gordon examined Julie’s isolated cells
by electron microscopy and found that the contractile cells
(<5 days in culture) resembled normal mature smooth muscle
with cytoplasm full of myofilaments, but that the broadened
and flattened cells 7 days in culture exhibited the same synthetic morphology as his regenerating smooth muscle cells
in vivo. Thus began our working collaboration of over 40
years—and our personal one resulting in 3 children and 2
(so far) grandchildren.
In the same 1974 article,3 we showed that if the cells had
been seeded densely in primary culture such that a confluent
monolayer resulted after ≈2 days of proliferation, then they
drew up into hills and valleys. Within a further 2 to 3 days, the
cells in the reaggregated hills returned to their original spindle/
elongated ribbon shape and recommenced spontaneous,
and often synchronous, contraction. Ultrastructurally, they
had regained myofilaments and lost many of their synthetic
organelles. This showed that the changes in the structure and
function of the mature smooth muscle cells in primary culture
were reversible. However, if the enzymatically isolated visceral
smooth muscle cells were seeded sparsely in primary culture,
then they underwent the same change in morphology as the
more densely seeded cells on days 5 to 7, took ≈3 weeks and
many more cell divisions to achieve confluence, and did not
revert to their original phenotype but appeared permanently in
a synthetic state.
Our studies also noted that a small percentage (≈0.05%)
of visceral smooth muscle cells in the first 2 to 4 days in
culture underwent a single division while still contracting
strongly, ceasing spontaneous contraction at the beginning
of prophase and resuming when the daughter cells were in
interphase.4 These cells were indistinguishable morphologically from other smooth muscle cells in the culture, and like
Smooth Muscle Phenotypic Change
The article by Wissler had a huge impact on the 2 of us. In
1970, we were PhD students studying different aspects of
smooth muscle at the University of Melbourne. Gordon was
structure (histology) while Julie was function (cell biology
and biochemistry). Gordon had noted that during regeneration
and repair following injury in vivo, smooth muscle cells in visceral organs, such as the vas deferens and taenia coli, lost their
myofilaments and gained large amounts of synthetic organelles (rough endoplasmic reticulum, Golgi, mitochondria, free
ribosomes) before proliferating2 (Figures 1 and 2). Julie had
noted that individual visceral smooth muscle cells enzymatically isolated and seeded in primary culture contracted spontaneously at a rate of 1× to 7× per minute, but that after 5
to 7 days in culture changed from spindle/elongated ribbon
to become broader and flatter, and then ceased contractions.3
This change in shape and behavior nearly always preceded
the cell’s commencement of proliferation in the presence of
From the Australian Institute for Bioengineering and Nanotechnology (J.H.C.) and School of Biomedical Sciences (G.R.C.), University of Queensland,
Brisbane, Queensland, Australia.
Correspondence to Julie H. Campbell, 757 South Branch Rd, Maryvale, Queensland 4370, Australia. E-mail [email protected]
© 2012 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org
1784
DOI: 10.1161/ATVBAHA.111.243212
Campbell and Campbell History of Discovery 1785
observed in mitosis are at least partially modulated toward
the synthetic state2 (Figure 3). Indeed, Poole et al6 stated the
following: “On looking at cells in the tunica media further
and further away from the site of injury, it was seen that there
was a continuous gradient in cell morphology from normal
smooth muscle cells of the tunica media (which presumably had not been injured) to the cells showing great lack of
differentiation nearer to the silk suture. Mitoses were seen
among these cells.”
Distinguishing Synthetic-State Smooth
Muscle From Fibroblasts
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Figure 1. Smooth muscle cells in the contractile state. Most of
their cytoplasm is filled with myofilaments. Transverse section
through the rabbit radial artery.
them gradually flattened and lost their contractile ability.
Division of smooth muscle with a contractile morphology
was observed in the developing chicken gizzard by Cobb and
Bennett,5 but most reports showed that the majority of cells
Figure 2. Synthetic-state smooth muscle cell from guinea-pig vas
deferens, 1 week after transplantation to the anterior eye chamber.
Note the large numbers of synthetic organelles and sparse peripheral bundles of myofilaments (Reproduced from Campbell et al2).
The close similarity in the appearance of fibroblasts and synthetic-state smooth muscle cells, both in culture and in developing and regenerating smooth muscle organs, was causing
considerable confusion in the literature during the 1960s and
1970s. However, with experience they could still be distinguished. Under phase-contrast microscopy, synthetic-state
smooth muscle cells are more phase-dense than fibroblasts and
have considerably fewer inclusions in their cytoplasm. The
nucleus has a more defined outline and is sausage shaped instead
of round or oval as in fibroblasts, and the nucleoli are less phasedense. Ultrastructurally, synthetic-state smooth muscle cells can
be distinguished by the presence of a complete basal lamina,
more plasmalemmal vesicles along the plasma membrane, and
larger bundles of thin filaments with associated dark bodies.
An easier way to distinguish the 2 cell types was found by
our collaborator Ute Groschel-Stewart, who developed antibodies to smooth muscle contractile proteins visualized with
fluorescein isothiocyanate. Her antibody to smooth muscle
myosin heavy chain did not cross-react with skeletal or cardiac
Figure 3. Dividing smooth muscle cell from guinea-pig vas
deferens, 1 week after transplantation to the anterior eye
chamber. Most of the cytoplasm is filled with synthetic organelles
(Reproduced from Campbell et al2).
1786 Arterioscler Thromb Vasc Biol August 2012
muscle, but strongly reacted with contractile-state smooth
muscle myofibrils and only weakly with fibroblasts at room
temperature. Reaction of the myosin antibody with smooth
muscle was greatly reduced in cells that had undergone phenotypic change to the synthetic state.7 The major break came
with Ute’s development of an antibody to native smooth
muscle actin, a feat that had eluded many other researchers.
This antibody reacted with smooth muscle cells irrespective of
their phenotype, but did not react with fibroblasts or endothelial cells.8 A definitive way of distinguishing synthetic smooth
muscle cells and fibroblasts was now available.
Phenotypic Change in Vascular Smooth Muscle
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All our early work was done using visceral smooth muscle
(vas deferens, taenia coli, ureter, gizzard) that contracted
spontaneously in the first few days of primary culture and
contained large amounts of myofilaments. If we had begun
our studies using vascular smooth muscle cells, which appear
morphologically less distinctly smooth muscle than their visceral counterparts, the changes that we so clearly saw may not
have been observed. However, when we specifically looked
for these changes in vascular smooth muscle cells in culture,
we saw them.9 Our article described a method for the growth
of large numbers of enzyme-isolated vascular smooth muscle
cells from adult humans, monkey, and rabbit in primary culture
and compared the properties of these cells with those that had
migrated from explants and those in subculture. Use of Ute
Groschel-Stewart’s antibodies not only showed that our cultures were consistently >99% pure smooth muscle but also that
the cells that had migrated from explants and those in subculture were already in the synthetic state—loss of the contractile
state generally being a prerequisite for migration and proliferation, and a permanent state after multiple rounds of cell division. We drew the conclusion that enzyme-isolated cells were
phenotypically more representative of normal, mature smooth
muscle cells in the artery wall and should be used instead of
explant outgrowth or subcultured cells if one were seeking to
discover the initiating events of neointima formation.
This work tended to be in direct conflict with that of Russell
Ross, who was using serially subcultured monkey aorta
smooth muscle (which had originally grown out of explants
over 4 weeks) to investigate the cell biology of atherosclerosis.
He showed that these cells, in the ninth subculture or later,
synthesize collagen and elastin, but he also maintained that
they exhibited a differentiated appearance under the electron
microscope with abundant myofilaments and dense bodies.10
However, we showed that sectioning the cells close to the
surface of the culture dish will give an erroneous picture,
because that is where large numbers of attachment filaments
and stress fibers are located, whereas the rest of cytoplasm
contains mainly synthetic organelles.11 Others variously
described the smooth muscle cells that grew from explants of
aortic tissue in culture as fibroblast-like or modified smooth
muscle cells, with relatively sparse myofilaments generally
located at the cell periphery. Indeed, Ross10 himself stated
that he presumed the filaments he saw were myofilaments
and that his aortic outgrowth and subcultured cells “markedly
resembled smooth muscle cells in vivo that are engaged in the
synthesis and secretion of extracellular proteins.”
We thus decided that to resolve our conceptual differences
with Ross, we should spend some time in his laboratory. We
both won postdoctoral traveling fellowships and worked 1
year (1976) in London, where we continued our collaboration with Ute Groschel-Stewart using her (then) very novel
antibodies, 9 months in the Department of Pharmacology at
the University of Iowa, Iowa City (where we were married in
March 1977), followed by 5 months in Seattle.
During those 5 months, we worked and wrote 2 articles with
Ross. The first showed that the change in the phenotype of
enzyme-isolated smooth muscle cells occurred in primary culture
irrespective of whether whole serum (normal or hyperlipidemic)
or platelet-deficient serum was present. It also clearly showed
a phenotype-dependent response to serum mitogens, with minimal proliferation occurring until the cells had undergone distinct
morphological changes characterized by loss of myofilaments
and more extensive synthetic organelles.12 Some years later, we
showed that smooth muscle cells, both densely seeded early in
primary culture and those sparsely seeded and proliferating, possess a relatively large number of binding sites for [125] plateletderived growth factor-BB averaging 126 fmol/106 cells with a
Kd of 0.53 nmol/L.13 The second article with Russell Ross was a
61-page invited review in Physiological Reviews, “The smooth
muscle cell in culture.”14 It was much more than a review and
contained a wealth of original data, showing that smooth muscle
cells can alter their structure and function in response to functional demands (eg, injury/repair). It emphasized that there
are not 2 phenotypic states, contractile and synthetic, but that
smooth muscle can exist in a spectrum of phenotypes, with contractile and irreversibly synthetic at the 2 ends and many forms
in between (hence the ability of some smooth muscle cells in
culture to divide while still contracting, as described in our 1974
article4). This collaborative article went a long way in winning
Ross over and in stimulating the use of smooth muscle culture as
a tool to investigate developmental and disease processes.
Reversibility of Phenotypic Change
In 1981, we showed that reversibility of smooth muscle phenotypic change was dependent on the number of cell doublings
of the population before confluency was achieved.15 Fewer
than 5 cell doublings allowed the cells to return to the contractile state (reversibly synthetic), whereas >9 cell doublings rendered them incapable of return (irreversibly synthetic), but not
senescent. However, our later studies showed that any return
to the contractile state in primary culture was not complete.
Although the volume fraction of myofilaments in the cytoplasm of the cells returned to their original levels, as did the
level of α-smooth muscle actin mRNA, the level of α-smooth
muscle actin protein, once decreased upon phenotypic change,
remained low, irrespective of the confluency or low population doublings.16 This suggests that an increase in the volume
fraction of myofilaments is not necessarily caused through an
increase in α-smooth muscle actin content, but may be caused
by polymerization of cytoplasmic actins or by an increase in
myosin or intermediate (10 nm) filaments.
Lipid Metabolism and Phenotype
We had returned to Melbourne, Australia, early in 1978, and our
first child was born in October 1978, the second in April 1980,
Campbell and Campbell History of Discovery 1787
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and the third in July 1981. Julie then decided to henceforth
publish as Campbell, rather than as Chamley-Campbell (or
Chamley). The presence of experts in lipid metabolism at the
Baker Medical Research Institute where we worked spurred
an interest in determining whether synthetic versus contractile
smooth muscle had a propensity to accumulate lipid. Indeed,
we showed that a change in smooth muscle phenotype to the
synthetic state is accompanied by distinct changes in the cells’
ability to metabolize low-density lipoprotein (LDL), with the
rate of 125I-labeled LDL degradation decreasing to about
one fifth of the level in contractile state cells.17 This was not
because of changes in the number or affinity of LDL receptors
on the cells because saturable binding of LDL was unaltered.
The specific activities of lysosomal enzymes acid phosphatase
and N-acetyl-β-glucosaminidase increased with change to the
synthetic state as did cytochrome c oxidase (mitochondria)
and nicotinamide adenine dinucleotide phosphate–dependent
cytochrome c reductase (endoplasmic reticulum), but a
decrease in the specific activity of the lysosomal enzyme acid
cholesterol esterase. Significantly, more 3H-cholesteryl oleate
was recovered in synthetic than contractile cells after incubation
with unlabeled LDL and 3H-sodium oleate. Similar but greatly
enhanced differential effects on binding, degradation, and lipid
accumulation by synthetic (particularly irreversibly synthetic)
versus contractile state cells were shown by incubation in
medium containing β-very low-density lipoprotein isolated
from hyperlipidemic serum,17 and the effect was further
exacerbated by incubation of the β-very low-density lipoprotein
with endothelial cells or macrophages that caused extensive
lipid peroxidation. Morphologically, the synthetic-state cells
became almost completely filled with lipid droplets, whereas the
contractile-state cells were unaffected. Similarly, it was shown
that lipid accumulation occurred within the (morphologically)
synthetic-state cells of intimal thickenings in the aortae of
rabbits fed a 1% cholesterol-enriched diet, whereas there was
no accumulation in contractile-state cells of the media.
Collagen/Glycosaminoglycan Synthesis
and Phenotype
We also showed that collagen synthesis increased significantly
upon change to the synthetic state and was greatest (35-fold) in
cells that had undergone >5 cumulative population doublings.18
Noncollagen protein synthesis also increased but to a much
lower extent. The increases in collagen and noncollagen protein were directly related to phenotype and independent of proliferation/quiescence. Type I collagen (as opposed to collagens
type III or V) was the predominant collagen synthesized by all
phenotypes, with a higher proportion synthesized by syntheticstate cells. Other studies showed that glycosaminoglycan synthesis increased 10-fold in synthetic- versus contractile-state
cells, with higher proportions of chondroitin sulfate A/C and
dermatan sulfate.19 Expression of an adhesion molecule for
leukocytes, intracellular adhesion molecule-1, occurred only
on synthetic-state smooth muscle, with no expression by contractile-state cells even after stimulation with interleukin-1β.20
Smooth Muscle Phenotypic Change In Vivo
Using ultrastructural morphometry, we showed that in forming a neointimal thickening after endothelial denudation,
smooth muscle cells in the underlying media underwent
a change in phenotype before their migration to the intima
and proliferation and that the change was reversed once re-­
endothelialization had occurred.21 However, if the segment
of endothelium experimentally removed from an artery was
small such that complete re-endothelialization occurs rapidly, an intimal thickening did not develop even though platelet aggregation and release of platelet-derived growth factor
occur within the first 24 hours.
About 10 years later, we showed that 3 days after ballooncatheter injury, the level of heparanase activity in the artery
wall is increased by ≈50% and by 140% at 2 weeks, returning
to control levels at 6 weeks.22 Matrix metalloproteinase activity followed a similar pattern. In a subsequent article, both
heparan sulfate and chondroitin sulfate were found in close
association with smooth muscle cells of the uninjured arterial media as well as being more widely spread within the
matrix. Within 6 hours after arterial injury, there was loss of
the regular pericellular distribution of both glycosaminoglycans, which was associated with a significant expansion of the
extracellular space. This preceded the change in phenotype of
the smooth muscle cells as observed with ultrastructural morphometry. The decrease in glycosaminoglycans was greatest
at 4 days, after which both rapidly returned around the cells of
the media, but the intimal cells failed to produce heparan sulfate as readily as they produced chondroitin sulfate. We also
showed that heparan sulfate proteoglycan extracted from the
artery wall inhibited the development of a neointima in balloon-injured rabbit carotid arteries when applied to the adventitia in a pluronic gel, as well as inhibiting phenotypic change
in vitro. Phosphomannopentaose sulfate (also called PI-88),
which inhibits the activity of heparanase, also prevented
a change in smooth muscle phenotype and reduced intimal
thickening after balloon injury of rat and rabbit arteries.23
In 1985, our postdoc Peter Mosse used quantitative morphometry to analyze the volume fraction of synthetic organelles in smooth muscle cells of diffuse intimal thickenings
of human carotid arteries adjacent to atherosclerotic plaques
taken at endarterectomy. He found enormous phenotypic
variability among these cells, but that the vast majority had
most of their cytoplasm filled with rough endoplasmic reticulum, free ribosomes, and mitochondria, equating to a 2-fold
increase in the volume fraction of synthetic organelles compared with smooth muscle cells of the subjacent media.24 In
a subsequent article, he showed no difference in the volume
fraction of synthetic organelles in atherosclerosis-free diffuse
intimal thickenings of human tissue taken at autopsy compared with the underlying media. These studies, together with
our studies showing lipid accumulation in synthetic but not
contractile-state cells, added further to our thesis that change
in smooth muscle phenotype was important in atherogenesis.25
Macrophages Induce a Change in
Smooth Muscle Phenotype
Our next discovery was that living macrophages induce a
change in the phenotype of smooth muscle cells in vitro and
then stimulate their rate of proliferation beyond confluency to
form multilayers. We also showed that macrophages have in
their lysosomes a heparan sulfate–degrading endoglycosidase
1788 Arterioscler Thromb Vasc Biol August 2012
that cleaves internal glycosidic bonds and that its action
is sufficient to induce a change in the phenotypic expression of smooth muscle cells.26 Our results further suggested
that macrophages possess sulfatases and exoglycosidases,
which sequentially release inorganic sulfates and monosaccharide residues from the nonreducing ends of the heparan
sulfate fragments released by the endoglycosidase. We, therefore, suggested that macrophages, as well as being a source
of lipid-laden foam cells in atherosclerosis, may play other
important roles in this disease, such as initiating change in
smooth muscle phenotype and influencing their proliferative
response and pattern of growth. Our results showing enhanced
proliferation were consistent with the findings of others that
macrophages produce a potent mitogen resembling plateletderived growth factor.
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How Might Degradation of Heparan Sulfate
Trigger a Change in Phenotype?
Cell-associated heparan sulfate proteoglycans occur as
membrane-intercalated glycoproteins where the core protein
is anchored in the lipid interior of the plasma membrane, and
the heparan sulfate chains bind to specific sites on collagen,
laminin, and fibronectin. The function of the proteoglycanmediated interaction is to promote the organization of actin
filaments in the attaching cell, which also has the effect of
stabilizing cell morphology; thus, removal and destruction of
cell-surface heparan sulfate at these sites may initiate a change
in smooth muscle phenotype through disorganization of actin
filaments with subsequent influences on gene expression. The
observation that trypsin (which releases the heparan sulfate
proteoglycans from the cell surface) does not by itself induce
a change in smooth muscle phenotype suggests that the
heparan chains must be completely destroyed or otherwise
removed from the vicinity of the cell for this to occur. The
ability of free heparin to prevent a change in the phenotype
of smooth muscle cells whose extracellular matrix and basal
lamina have been degraded and removed during enzymatic
isolation supports this view. We showed that smooth muscle
cells in the contractile state continuously internalize and
degrade their own surface heparan sulfate to free sulfate, and
that this occurs via a nonlysosomal pathway.26 Thus, heparan
sulfate internalized from the cell surface may play a role in
maintaining the smooth muscle cells in the contractile state.
In other cell systems, it is known that surface heparan sulfate
is regularly internalized and degraded, whereas fractions
enriched in the rare 2-O-sulfate glucuronate units are not fully
degraded but transported to the nucleus where they influence
gene expression.
However, over the years the factors reported to control
smooth muscle phenotype have been many and complex. We
have shown that T lymphocytes, T-lymphocyte–conditioned
medium, and a T-lymphocyte–derived cytokine, interferon-γ,
are potent inducers of smooth muscle phenotypic change.27
Activation of RhoA (a key regulator of the actin cytoskeleton) by sphingosine-1-phosphate enhanced the expression
of contractile proteins α-smooth muscle actin, smooth muscle myosin heavy chain, and SM-2, whereas inhibition of
RhoA induced a more extreme synthetic phenotype including
increased expression of vimentin.28 Transient transfection
of synthetic-state cells with the constitutively active RhoA
(Val4RhoA) caused a reduction in cell size and reorganization of cytoskeletal proteins to resemble that of the contractile
phenotype. Others have reported that oxidized phospholipids
or unsaturated lysophosphatidic acids (which activate Rho)
induce phenotypic change. Platelet-derived growth factor-BB
was reported to induce smooth muscle phenotypic change in
vitro, specifically through destabilization of α-actin mRNA,
with interleukin-1β playing a synergistic role. Various other
factors have been reported.
Perspectives
Over the past 20 or so years, many other researchers have
contributed to our knowledge of smooth muscle phenotype.
In particular, they have investigated the complex mechanisms
that regulate transcription of smooth muscle gene expression
and control smooth muscle differentiation and phenotypic
modulation, and identified a number of important marker
genes.29–32 They have also shown that smooth muscle phenotypic modulation involves the activation of micro RNAs and
embryonic stem cell pluripotency genes and that epigenetic
mechanisms play an important role. These are important and
exciting discoveries that have greatly advanced the field.
However, as is often the case in science, the originators of
the ideas on which much of this work depends appear to have
been forgotten. Rarely is there acknowledgment of the studies of Wissler and others who first observed the multifunctional mesenchyme nature of smooth muscle or of ours that
described its phenotypic modulation in response to functional
demands, cataloged its changes in structure and function,
examined its controlling factors, and highlighted its relevance
to disease processes such as atherosclerosis. Unfortunately,
once articles, particularly major reviews, appear that lack
historical perspective of discovery, the wheel becomes reinvented, and others adopt the same citations, albeit in otherwise excellent articles.
This loss of historical perspective regarding smooth muscle
phenotypic modulation is what the current article has tried to
mitigate. It also serves as a reminder that the basic cell biology
related to phenotypic change should not be forgotten among
studies of transcriptional regulators.
Sources of Funding
This work was supported by the National Health and Medical
Research Council of Australia.
None.
Disclosures
References
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